Identification of a G protein-coupled receptor specifically responsive to β-alanine

Abstract

We isolated a cDNA encoding an orphan G protein-coupled receptor, TGR7, which has been recently reported to correspond to MrgD. To search for ligands for TGR7, we screened a series of small molecule compounds by detecting the Ca2+ influx in Chinese hamster ovary cells expressing TGR7. Through this screening, we found that β-alanine at micromolar doses specifically evoked Ca2+ influx in cells expressing human, rat, or mouse TGR7. A structural analogue, γ-aminobutyric acid, weakly stimulated cells expressing human or rat TGR7, but another analogue, glycine, did not. In addition, β-alanine decreased forskolin-stimulated cAMP production in cells expressing TGR7, suggesting that TGR7 couples with G proteins G q and Gi. In guanosine 5′-O-3-thiotriphosphate binding assays conducted using a membrane fraction of cells expressing TGR7, β-alanine specifically increased the binding of guanosine 5′-O-3-thiotriphosphate. When a fusion protein composed of TGR7 and green fluorescent protein was expressed in cells, it localized at the plasma membrane but internalized into the cytoplasm after treatment with β-alanine. In addition, we found that β-[3H]alanine more efficiently bound to TGR7-expressing cells than to control cells. From these results, we concluded that TGR7 functioned as a specific membrane receptor for β-alanine. Quantitative PCR analysis revealed that TGR7 mRNA was predominantly expressed in the dorsal root ganglia in rats. By in situ hybridization and immunostaining, we confirmed that TGR7 mRNA was co-expressed in the small diameter neurons with P2X3 and VR1, both in rat and monkey dorsal root ganglia. Our results suggest that TGR7 participates in the modulation of neuropathic pain.